Kyushu Dental University

Bmi1 is a polycomb protein localized in stem cells and maintains their stemness. This protein is also reported to regulate the expression of various differentiation genes. In this study, to analyze the role of Bmi1 during dentinogenesis, we examined the immunohistochemical localization of Bmi1 during rat tooth development as well as after cavity preparation. Bmi1 localization was hardly detected in the dental mesenchyme at the bud and cap stages. After the bell stage, however, this protein became detectable in preodontoblasts and early odontoblasts just beginning dentin matrix secretion. As dentin formation progressed, Bmi1 immunoreactivity in the odontoblasts decreased in intensity. After cavity preparation, cells lining the dentin and some pulp cells under the cavity were immunopositive for Bmi1 at 4 days. Odontoblast-like cells forming reparative dentin were immunopositive for Bmi1 at 1 week, whereas their immunoreactivity was not detected after 8 weeks. We further analyzed the function of Bmi1 using KN-3 cells, a dental mesenchymal cell line. Overexpression of Bmi1 in KN-3 cells promoted mineralized tissue formation. In contrast, siRNA knockdown of Bmi1 in KN-3 cells reduced alkaline phosphatase activity and the expression of odontoblast differentiation marker genes such as Runx2, osterix, and osteocalcin. Additionally, KN-3 cells transfected with siRNA against Bmi1 showed reduced nuclear transition of β-catenin and expression of phosphorylated-Smad1/5/8. Taken together, these findings suggest that Bmi1 was localized in the odontoblast-lineage cells in their early differentiation stages. Bmi1 might positively regulate their differentiation by accelerating Wnt and BMP signaling pathways.

Titanium implants are widely utilized for tooth root reconstruction due to their excellent mechanical and biological properties. However, their mechanical properties differ from those of dentin. This study aims to develop a 3D-printable polymer-infiltrated ceramic network (PICN) as a tooth root restoration material that mimics the natural root shape, mechanical properties, and biocompatibility. A bioactive glass (BG)-based photocurable slurry was prepared for vat photopolymerization, printed, sintered, and subsequently polymer-infiltrated to form 3D-printable PICN (3D-PICN). For comparison, 3D-printable BG (3D-BG), a dense ceramic without resin infiltration, was fabricated using the same printing and sintering process. The photocurable slurry was characterized for its rheological and photopolymerization behaviors, including viscosity, cure depth, degree of conversion, overgrowth, and printing accuracy. The results confirmed its suitability for vat photopolymerization, enabling the precise fabrication of tooth root-shaped structures. Mechanical properties, including work of fracture, flexural strength, flexural modulus, and Vickers hardness, were evaluated for both 3D-PICN and 3D-BG. The results revealed that the mechanical properties of 3D-PICN closely match those of dentin, whereas 3D-BG exhibits properties similar to enamel. Biocompatibility was assessed through in vitro simulated body fluid immersion tests and in vivo implantation in a ten-week-old rat tibia model, followed by histological analysis. The findings confirmed good biocompatibility of 3D-PICN with bone tissue. The 3D-PICN demonstrated excellent printability, mechanical compatibility with dentin, and favorable biocompatibility, suggesting its potential as a promising material for tooth root reconstruction applications.

Human dental pulp stem cells (hDPSCs) are a promising cell source for tooth regeneration therapies. However, conventional culture scaffold materials are often animal-derived, leading to immunogenicity concerns and limited availability. In this study, we explored phosphorylated cellulose nanofibers (P-CNFs), which have a fine fiber morphology and phosphate groups, as a novel scaffold material for cell culture. Immortalized hDPSCs were cultured on P-CNF scaffolds with different phosphate contents (0-1.42 mmol g^-1) prepared by varying the molar ratio of urea and diammonium hydrogen phosphate and the reaction time. Cells cultured on unmodified CNFs exhibited poor adhesion and formed spheroids, indicating low bioadaptability. In contrast, P-CNF scaffolds with moderate phosphate content (0.54-0.78 mmol g^-1) significantly improved cell adhesion; further increases in phosphate content decreased cell adhesion, indicating a strong dependence on phosphate content. Intriguingly, even in the absence of differentiation inducers, hDPSCs on P-CNF scaffolds with an optimal phosphate content of 0.78 mmol g^-1 showed equal or higher expression of hard tissue marker genes compared to collagen scaffolds with differentiation inducers, suggesting that P-CNFs can directly promote hard tissue differentiation. These findings highlight plant-derived, animal-free P-CNFs as a promising biomaterial for advanced dental tissue engineering.